PROJECTSUMMARY/ABSTRACT Everyyear,>1millionpatientsundergobonerepairproceduresintheUnitedStates.Autologousbonegrafting remainsthepreferredtreatmentforbonedefects,butthispracticeislimitedbyboneavailabilityanddonorsite morbidity from harvesting the bone. Alternatively, the development of therapies that exploit the osteogenic potential of bone marrow-derived mesenchymal stem cells (bmMSCs) continues to be a priority in osteoregenerative medicine. However, efforts remain largely empirical due to poor understanding of the mechanismsregulatingbmMSCengraftmentandosteogenicactivityinvivo.Ourlong-termgoalistodevelopa regenerative therapy that is based on bioengineering an osteoinductive niche for human bmMSCs. We have foundthattheinvivopreservationofhumanbmMSCosteogenicpotentialdependsonsustainingproximityto endothelialcells(ECs)andonthetimelyengraftmentofbmMSCsasperivascularcells(Linetal.,PNAS2014). WehavealsofoundthatvascularnetworksbioengineeredusinghumaniliaccresttrabecularboneECs(bECs) couldspontaneouslyinduceosteogenicdifferentiationofbmMSCsatectopicsites.Incontrast,ECsfromother human tissues could not. In addition, we have identified five candidate genes (BMP2, BMP7, NOG, KITLG, MSX1)differentiallyupregulatedinbECs.Ouroverarchinghypothesisisthatbioengineeredmicrovesselslined withbECsserveasstablenichesforbmMSCsandautonomouslydriveosteogenesisviaregulationofspecific osteoinductivegenes.Moreover,wepostulatethatinducedpluripotentstemcells(iPSCs)couldofferaplentiful source of surrogate bECs, eliminating the need for harvesting autologous trabecular bone. To test these hypotheses and to elucidate the precise osteoinductive factors whereby human trabecular bECs uniquely regulateosteogenesis,weproposethreeSpecificAims.InAim-1,wewillbioengineervascularnetworkswith humanbECsandbmMSCsanddeterminethecapacitytoregeneratecritical-sizedorthotopicbonedefects.In Aim-2, we will determine the factors responsible for the unique in vivo osteoinductive potential of human trabecular bECs. We will knockout each candidate bEC gene and will determine the effect on in vivo osteogenesis.Tothisend,wewillusealuciferase-reporterdrivenbythehumanosterixpromotertomeasure bmMSCosteogenicactivityviabioluminescence.InAim-3,wewilldetermineconditionstogeneratesurrogate bECsfromiPSCs.WewillexaminewhetheriPSC-derivedECs(iECs)acquireosteoinductivepropertiesupon transplantationintobonesitesandareinturnabletoautonomouslyregulatetheosteogenicactivityofbmMSCs in vivo. We will use our murine calvarial bone defect model to determine the extent of in vivo osteogenic education of iECs by measuring (i) transcriptional profile modifications (RNAseq) and (ii) osteoinductive propertiesinengraftediECs.Wewillalsodeterminethelong-term(16weeks)bonerepaircapabilityofconstructs containingiECsandwhetherimplantingpre-educatediECsimprovestheextentofbonerepair.Collectively,we envisionthisresearchcouldbecomethebasisforanewstrategyintherepairofbonedefects.